New tools for anaerobic bacteria: metabolically engineered strains to improve energy supply, recombinant production of value-added products, and fluorescent reporter proteins

Abstract

Anaerobic bacteria are promising biocatalysts for producing industrially relevant high-value chemicals from various non-food feedstocks. While solventogenic clostridia are excellent alcohol producers, acetogenic bacteria have the distinction of being able to utilize C1 carbon sources and can thus help reduce greenhouse gas emissions. While several anaerobes are genetically accessible, various genetic tools are not yet established. In this work, new molecular tools for anaerobes are presented. This includes the establishment of heterologous pathways to provide additional ATP and to produce the biocommodities butanol and acetone. Genes of the arginine deiminase pathway were heterologously expressed in Acetobacterium woodii to overcome energetic limitations during autotrophic growth since the conversion of arginine to ornithine, ammonia, and CO2 provides ATP and finally boosts growth of recombinant A. woodii strains. The enhanced energy supply by the arginine deiminase pathway opens the door for the establishment of new recombinant pathways that require huge amounts of ATP. In addition, the application of the fluorescence-activating and absorption-shifting tag (FAST) as oxygen-independent fluorescent reporter protein further extended the molecular toolbox of anaerobes. As it turned out, FAST is perfectly suited for the construction of fluorescent FAST-tagged fusion proteins without negatively affecting the stability and functionality of the native enzyme. Both, the bifunctional aldehyde/alcohol dehydrogenase AdhE2 and the acetoacetate decarboxylase Adc from C. acetobutylicum were FAST-tagged and respective genes expressed in Eubacterium limosum. As a result, recombinant E. limosum strains produced 0.6 mM butanol and 1.6 mM acetone from the fluid C1 carbon source methanol, while production of FAST-tagged fusion proteins was tracked regarding fluorescence intensity during growth. In addition, the orthogonal green and red fluorescence of mutated FAST versions, greenFAST and redFAST, were evaluated and used for multicolor approaches under anaerobic conditions. Hence, the co-existence and dynamics in an anaerobic microbial co-culture consisting of two engineered C. saccharoperbutylacetonicum strains producing respective fluorescent proteins were monitored. Moreover, a tightly regulated inducible two-plasmid system based on the tcdR-PtcdB promoter system of C. difficile was constructed. The orthogonal green and red fluorescence of greenFAST and redFAST was used to confirm the co-existence of both plasmids in C. saccharoperbutylacetonicum during growth at single-cell level.